Ca2+/cation antiporters (CaCAs) are the major secondary Ca2+ transporter proteins in the plasma membrane. They play essential roles in many important Ca2+-mediated biological processes including cardiac contraction and neuronal transmission. In mammalian CaCA proteins, Ca2+ transport through their transmembrane domain is tightly controlled by their intracellular regulatory domain. However molecular mechanisms underlying Ca2+ transport and regulation of CaCA proteins are poorly understood due to the absence of an atomic structure of any member of the family. We have designed a strategy to elucidate these two important and interrelated mechanisms by structural and functional studies: 1) to elucidate the Ca2+ transport mechanism, we have crystallized the YfkE protein, a prokaryotic Ca2+ transporter and CaCA homolog sharing conserved membrane topology and sequence with mammalian CaCAs. We have obtained crystals diffracting to 6 resolution, and have designed innovative approaches to optimize the crystallization for structure determination by x-ray crystallography. The atomic structure of YfkE protein will not only provide the first structural basis for analyzing the Ca2+ transport mechanism of CaCAs, but also offers the first opportunity to understand in structural terms the Ca2+ selectivity and conductivity essential for Ca2+ homeostasis; 2) We have found that the Ca2+ transport activity of the YfkE protein is coupled with phosphate anion co-transport, a previously unrecognized aspect of a CaCA mechanism. We will analyze the Ca2+/phosphate co- transport pathway by mutagenesis in inside-out vesicles. We will test whether Ca2+/phosphate co- transport occurs in other CaCA proteins. These studies will provide the first data on Ca2+/phosphate co- transport and provide insight to phosphate involvement in Ca2+ homeostasis; 3) to elucidate the regulatory mechanism of mammalian CaCA proteins, our preliminary structural studies with Drosophila CaCA protein CALX suggest that the regulation is achieved by subdomain conformational changes induced by Ca2+ and Na+ interactions in the intracellular regulatory domain. To test this hypothesis, we will determine structures of the intracellular domain of CALX and examine the regulatory mechanism by mutagenesis and electrophysiology. In addition, by combining the structures of the prokaryotic Ca2+ transporter YfkE and the eukaryotic regulatory domain of CALX, we will be able to generate the first structural model to understand Ca2+ transport and regulatory mechanisms of the important CaCA proteins.